Production and applications for polyvalent vaccines against diseases caused by papilloma viruses

ABSTRACT

A vaccine against disease caused by papilloma viruses is described in certain embodiments, as well as certain vectors, obtainable by the following methods: (a) one or more expression vectors that contain the DNA code for a structural protein of papilloma viruses or a fragment thereof are injected into mammals, whereby in at least some of the expression vectors randomly generated heterologous sequences are inserted into the DNA code (b) serums are obtained from the mammals and these are tested for the presence of antibodies against particles of various papilloma virus types, and (c) using the serums tested, the structural protein gene clones are identified that code for a polyvalent vaccine, and (d) the vaccine is produced from them. Procedures for producing a vaccine is also described, together with its use for vaccination against diseases caused by papilloma viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of U.S. patent applicationSer. No. 10/485,454, filed Jan. 30, 2004, entitled “Polyvalent VaccineAgainst Diseases Caused By Papilloma Viruses, Method For The ProductionAnd The Use Thereof” which is a U.S. national stage application filedunder 35 U.S.C. § 371 and claims priority to PCT/EP02/08360 (WO03/011335), internationally filed Jul. 26, 2002, which claims priorityto DE 101 37 102.0, filed Jul. 30, 2001, priority to all of which areclaimed and which are hereby incorporated by reference herein.

FIELD OF USE

Some aspects of certain embodiments of the invention relate topolyvalent antibodies or vaccines against diseases caused by papillomaviruses, as well as the production process and application of theantibodies or vaccines.

BACKGROUND

Papilloma viruses are a family with considerably more than 80 genotypes.Infection with papilloma viruses can lead to warts, papillomas,acanthomas, and skin and cervical carcinomas. A single illness can becaused by various papilloma virus types.

The capsids of the individual types of human pathogenic papillomaviruses (HPV) differ in their antigen characteristics (epitopes),meaning that after immunization with a specific HPV type, neutralizingantibodies cannot be induced against capsids of other HPV types.However, such antibodies would be necessary to give comprehensiveprotection against diseases that can be caused by different HPV types.

An example is that infection with one of more than ten different HPVtypes can lead to cervical cancer. Although the virus particles of theindividual types are very similar in their composition, they carrydifferent neutralizing epitopes on their surface and are therefore onlyrecognized by the immune system if there has been either a previousnatural infection or vaccination with particles of the same type, andtype-specific (neutralizing) antibodies are induced.

Vaccines for the effective prevention of diseases caused by HPV shouldtherefore contain a mixture of various virus types in order to givecomprehensive protection. The production of such vaccines is, however,rendered more difficult owing to the fact described above, namely thatone and the same disease can be caused by different HPV types.

To date only monovalent HPV vaccines have been developed, in other wordsvaccines directed against only one HPV type. However these have theserious disadvantage that they only guarantee protection against thisone special HPV type, and not against other HPV types. Thus, monovalentHPV vaccines do not furnish a comprehensive immune reaction. Moreover,the production of conventional vaccines against HPV typically requiresthe production and purification of L1.

SUMMARY

Consequently the present invention is based on making a vaccine and alsoa process for its simple production available with which an immuneresponse against different HPV types can be obtained. By constructinglibraries of one type of HPV antigenic biomolecule in which randompeptide sequences are inserted, the resulting proteins of some of theclones in the libraries are able to induce neutralizing antibodiesagainst at least two HPV types (e.g., HPV 16, 18, 31, and 45).

The screening of a library of such clones, however, is challenging usingconventional techniques. Conventional techniques for screening such alibrary would involve a step of production and purification of L1. Thisstep, however, may be eliminated by using DNA immunization. Further,many clones may be simultaneously screened by examining sera from ananimal that has been inoculated with a plurality of DNA clones.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of experimental data wherein mice (three per group)were immunized with different amount ratios of HPV 16 L1 DNA and emptypUF3 vector. The sera of the mice were tested in VLP-based ELISA for thepresence of anti-HPV 16 antibodies.

FIG. 2 is a plot of experimental data wherein mice (three per group)were immunized with different amount ratios of HPV 16 L1 and GFP DNA.The sera of the mice were tested in VLP-based ELISA for the presence ofanti-HPV 16 antibodies.

FIG. 3 is a plot of experimental data wherein mice (three per group)were immunized with different amount ratios of HPV 16 L1 DNA and HPV 18L1 DNA. The sera of the mice were tested in VLP-based ELISA for thepresence of anti-HPV 16 antibodies.

FIG. 4 is a plot of experimental data wherein mice (three per group)were immunized with different amount ratios of HPV 16 L1 and HPV 18 L1DNA. The sera of were tested in VLP-based ELISA for the presence ofanti-HPV 18 antibodies.

FIG. 5 depicts a combinatorial process for generating an antibody and/ora DNA clone that generates an antibody against a plurality of papillomavirus (PV) types.

DETAILED DESCRIPTION

By constructing libraries of one type of HPV antigenic biomolecule(e.g., HPV 16 L1) in which random peptide sequences are inserted intosurface loops, the resulting proteins of some of the clones in thelibraries are able to induce neutralizing antibodies against more thanone HPV type (e.g., both HPV 16 and HPV 18).

The antibodies are useful as diagnostic agents for identification of PV,as research reagents, and as markers. For example, antibodies that aregeneric to PV or to a subset of HPVs may be used to identify PV or asubtype of PV. Scientists commonly use antibodies as tools to identify,inhibit, or label molecules, and a large market for such research toolsexists.

DNA clones that elicit an immunogenic response in an animal, e.g.,production of an antibody, may be used directly in an animal as avaccine. Moreover, such clones may be used as research tools. Forexample, a DNA fragment that encodes an antigen for a plurality of HPVswould be useful for generating antibodies that recognize a plurality ofHPVs. And such clones could be used to infect test cells or animals tocreate a condition wherein they expressed multiple HPV antigens.

A library derived from an HPV antigenic biomolecule (e.g., HPV 16 L1) inwhich random peptide sequences are inserted into surface loops is usefulfor screening for antigens that are used to create antibodies againstHPV, HPV types (e.g., HPV 16 or HPV 18), and against multiple types ofHPV (e.g., both HPV 16 and HPV 18). Alternatively, other types of HPVmay be used instead of HPV 16, as will be evident to a person ofordinary skill after reading this disclosure. One specific region of asurface loop of the L1 protein is described below. In general, surfaceloop regions can be estimated from protein computational algorithms.However, a person of ordinary skill in the art can find surface loopregions based on information in the literature, computational estimatesof protein structure and/or empirical evaluation of the resultingantibodies using the approaches described herein using routineexperimentation.

A library may be referred to as having members that are clones. Clonesare members of a library that have common structural features, butdiffer from each other with regards to some structural feature. Clones,for example, may be expression vectors having an expression cassettethat each encodes a different DNA sequence. It is recognized that somemethods of preparing a library may result in some duplicate clones.Nonetheless, since each clone is considered to be distinct from theother, a plurality of clones refers to two clones that arenon-identical.

Vaccines and antibodies against a particular type of PV or HPV have beenreported, for example, in WO 03078455A3 WO 0045841, WO 0114416A3, WO9844944, WO 9531532 WO 9302184, U.S. Pat. Nos. 6,649,167 6,358,744,6,251,678, 6,245,568, 6,221,577, 6,183,745, 6,066,324, 6,025,163,5,888,516, 5,866,553, 5,840,306, 5,821,087, and 5,820,870 all of whichare hereby incorporated herein by reference. Further, sequence datainformation and identities for various PVs and HPVs are available inpublic databases accessible to persons of ordinary skill in these arts;for example U.S. Pat. No. 5,981,173 sets forth sequence data for HPV L1,L2, E6, and E7, NCBI accession No. X67161 sets forth a complete L1 DNAsequence, as per Longuet et al J. Clin. Microbiol. 34 (3), 738-744(1996); the HPV 16 complete genome sequence is set forth at NCBIaccession No. NC 001526 as per Kennedy et al., J. Virol. 65 (4),2093-2097 (1991); the HPV 18 complete sequence is set forth at NCBIaccession No. NC 001357 as per Cole et al., J. Mol. Biol. 193 (4),599-608 (1987). Moreover, certain HPV vaccines have been tested inhumans. Since vaccines for PV and HPV have been produced, tested, anddescribed elsewhere, a person of ordinary skill, after reading thisdescription, will be able to use the disclosed materials and methods tocreate vaccines.

One embodiment is a vaccine against diseases caused by papillomaviruses, obtainable by the following method:

(a) One or more expression vectors are injected into mammals. Thesevectors contain the DNA code for a structural protein of papillomaviruses (PV) or a fragment thereof, whereby in the case of at least someof the expression vectors randomly generated heterologous sequences areinserted into the DNA code.

(b) serums are obtained from the mammals and these are examined for thepresence of antibodies against particles of different papilloma virustypes.

(c) using the serums examined, the structural protein clones,particularly L1 clones, are identified that code for a polyvalentvaccine and

(d) the vaccine is produced from them.

Referring to FIG. 5, an embodiment of a method for using a combinatorialprocess to rapidly screen a large number of nucleic acid clones, e.g.,DNA clones, is depicted. Animals A1, A2, A3 . . . . An each receive aportion of library 100 of DNA clones, with the library having membersthat comprise nucleic acid expression vectors and/or expressioncassettes that encode potentially antigenic sequences to elicit animmune response from an animal. The library 100 is divided into a numberof groups that corresponds to the number of animal groups An, e.g., x,y, and z for three groups of animals. Each group x, y, z, has aplurality of components, e.g., 1, 2, 3 . . . n components. Each group isinjected into a single animal. As shown in FIG. 5, animal (or animalgroup) A1 receives x1, x2, x3 . . . xn clones of library 100, animalgroup A2 receives y1, y2 . . . y3 clones of library 100, and An receivesz1, z2 . . . z3 clones of library 100. DNA clones in library 100comprise expression control sequences 300 operably linked to thesequences that are to be expressed in the animal 302, 303, 304, 305,306, 307, 308, 309, 310, 311, 312, 313. Serum from an animal is screenedfor the presence of a desired immune response. An antibody and/or a DNAclone encoding an antigen that elicits a desired immunogenic responsefrom an animal is identified.

Library 100 may have a plurality of members that are each a nucleic acidclone that encode potentially antigenic sequences to elicit an immuneresponse from an animal. The clones may encode portions of a PV or HPVvirus, e.g., HPV 16, HPV 18, or any other HPV that is known. Asdescribed herein, portions of the DNA clones may include randomlygenerated sequences. The randomly generated sequences may be disposed inor near an known epitope for the HPV. As set forth herein, L1 loopportions include known epitopes for HPVs.

Animals 200, 202, 204 may be any animal that is capable of generating animmune response. For example, animals that have previously been adaptedfor laboratory uses may be used, including mice, rats, rodents, sheep,cows, calves, goats, dogs, monkeys.

Methods for screening animals for a target molecule having a particularproperty are known to those of ordinary skill in these arts. ELISA,dot-blot, gel electrophoresis, radiolabeling, immunolabeling, and aplethora of methods for identifying a target molecule are known in thebiological sciences. The serum from an animal may be screened for thepresence of antibodies by taking advantage of the specific binding of anantibody to a putative target, e.g., to a set of HPV types.Alternatively, other fluids or tissues from the animals may be screened.

The production of antibodies may be accomplished using methods known toartisans of ordinary skill. Polyclonal antibodies may conventionally beobtained from the animal's blood or serum and isolated by weight using acentrifuge and standard purification techniques. Monoclonal antibodiesmay conventionally be produced using hybridoma cell culture techniques.

The expression “fragments thereof,” as used above, indicates that theDNA codes for a protein that is shorter than the wild-type proteins, butwhich has the characteristics needed for this invention, especially thechemical, physical and/or functional characteristics.

Nucleic acids can be incorporated into vectors. Vectors may beexpression vectors containing an expression cassette, which is aninserted nucleic acid segment that is operably linked to an expressioncontrol sequence. A vector may be a replicon, e.g., a plasmid, phage, orcosmid, into which another nucleic acid segment may be inserted so as tobring about replication of the inserted segment. An expression vector isa vector that includes one or more expression control sequences, and anexpression control sequence is a DNA sequence that controls andregulates the transcription and/or translation of another DNA sequence.Expression control sequences include, for example, promoter sequences,transcriptional enhancer elements, and any other nucleic acid elementsrequired for RNA polymerase binding, initiation, or termination oftranscription. With respect to expression control sequences, the termoperably linked means that the expression control sequence and theinserted nucleic acid sequence of interest (also referred to herein asthe exogenous nucleic acid sequence that is intended to be expressed,also referred to as the exogenous nucleic acid sequence) are positionedsuch that the inserted sequence is transcribed (e.g., when the vector isintroduced into a host cell). A transcriptional unit in a vector maythus comprise an expression control sequence operably linked to anexogenous nucleic acid sequence. For example, a DNA sequence is operablylinked to an expression-control sequence, such as a promoter when theexpression control sequence controls and regulates the transcription andtranslation of that DNA sequence. The term operably linked includeshaving an appropriate start signal (e.g., ATG) in front of the DNAsequence to be expressed and maintaining the correct reading frame topermit expression of the DNA sequence under the control of theexpression control sequence to yield production of the desired proteinproduct. Examples of vectors include: plasmids, adenovirus,Adeno-Associated Virus (AAV), Lentivirus (FIV), Retrovirus (MoMLV), andtransposons.

There are a variety of promoters that could be used including, e.g.,constitutive promoters, tissue-specific promoters, and induciblepromoters. Promoters are regulatory signals that bind RNA polymerase ina cell to initiate transcription of a downstream (3′-direction) codingsequence.

Many different types of vectors are known. For example, plasmid vectorsand viral vectors, e.g., retroviral vectors, are known. Mammalianplasmid expression vectors typically have an origin of replication, asuitable promoter and optional enhancer, and also any necessary ribosomebinding sites, a polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. In addition, the expression vectors preferably contain a geneto provide a phenotypic trait for selection of transformed host cellssuch as neomycin resistance for eukaryotic cell culture, or tetracyclineor ampicillin resistance in E. coli. Retroviral vectors, which typicallytransduce only dividing cells, can be used. Adenoviral vectors, capableof delivering DNA to quiescent cells can be used. Another viral vectorsystem with potential advantages is an adeno-associated viral vector.

As used herein, the term nucleic acid refers to both RNA and DNA,including cDNA, genomic DNA, synthetic (e.g., chemically synthesized)DNA, as well as naturally occurring and chemically modified nucleicacids, e.g., synthetic bases or alternative backbones. A nucleic acidmolecule can be double-stranded or single-stranded (i.e., a sense or anantisense single strand). An isolated nucleic acid refers to a nucleicacid that is separated from other nucleic acid bases that are present ina genome, including nucleic acids that normally flank one or both sidesof a nucleic acid sequence in a vertebrate genome (e.g., nucleic acidsthat flank a gene). The term isolated as used herein with respect tonucleic acids also includes non-naturally-occurring nucleic acidsequences, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in a naturallyoccurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedat least one of the nucleic acid sequences normally found flanking thatDNA molecule in a naturally-occurring genome is removed or absent. Thus,an isolated nucleic acid includes, without limitation, a DNA moleculethat exists as a separate molecule (e.g., a chemically synthesizednucleic acid, or a cDNA or genomic DNA fragment produced by PCR orrestriction endonuclease treatment) independent of other sequences aswell as DNA that is incorporated into a vector, an autonomouslyreplicating plasmid, a virus (e.g., a retrovirus, lentivirus,adenovirus, or herpes virus), or into the genomic DNA of a prokaryote oreukaryote. In addition, an isolated nucleic acid can include anengineered nucleic acid such as a DNA molecule that is part of a hybridor fusion nucleic acid. A nucleic acid existing among hundreds tomillions of other nucleic acids within, for example, cDNA libraries orgenomic libraries, or gel slices containing a genomic DNA restrictiondigest, is not considered an isolated nucleic acid because such sourcesdo not indicate a role for the nucleic acid or its uses. Indeed, thereis often no knowledge of the sequences present in such sources untiltheir presence is hypothesized as a result of using hindsight in lightof a new sequence.

Examples of delivery of various embodiments as set forth herein, e.g.,nucleic acids, vaccines, antibodies, and vectors, include via injection,including intravenously, intramuscularly, or subcutaneously, and in apharmaceutically acceptable carriers, e.g., in solution and sterilevehicles, such as physiological buffers (e.g., saline solution orglucose serum). The embodiments may also be administered orally orrectally, when they are combined with pharmaceutically acceptable solidor liquid excipients. Embodiments can also be administered externally,for example, in the form of an aerosol with a suitable vehicle suitablefor this mode of administration, for example, nasally. Further, deliverythrough a catheter or other surgical tubing is possible. Alternativeroutes include tablets, capsules, and the like, nebulizers for liquidformulations, and inhalers for lyophilized or aerosolized agents.

Many known methods for delivering molecules in vivo and in vitro,especially small molecules, nucleic acids or polypeptides, may be usedfor embodiments described herein. Such methods include microspheres,liposomes, other microparticle vehicles or controlled releaseformulations placed in certain tissues, including blood. Examples ofcontrolled release carriers include semipermeable polymer matrices inthe form of shaped articles, e.g., suppositories, or microcapsules andU.S. Pat. Nos. 5,626,877; 5,891,108; 5,972,027; 6,041,252; 6,071,305,6,074,673; 6,083,996; 6,086,582; 6,086,912; 6,110,498; 6,126,919;6,132,765; 6,136,295; 6,142,939; 6,235,312; 6,235,313; 6,245,349;6,251,079; 6,283,947; 6,283,949; 6,287,792; 6,296,621; 6,309,370;6,309,375; 6,309,380; 6,309,410; 6,317,629; 6,346,272; 6,350,780;6,379,382; 6,387,124; 6,387,397 and 6,296,832. Moreover, formulationsfor administration can include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids, and powders.

When producing the vaccine according to the invention, the gene codingfor PV capsids of a specific type, for example L1, can therefore bemodified by inserting randomly generated sequences. Without priorproduction and cleaning of the capsids, for example by expression of theL1 gene using recombinant vectors such as plasmids, serums are producedthrough immunization with several L1 expression vectors that can bedefined as pools of expression vectors, and these serums are then testedfor reactivity with capsids of different PV types. After this, the poolsmay be isolated, and in this way the capsids with cross-neutralizingepitopes identified.

Persons of ordinary skill in these arts will be able to determine if anucleotide sequence encodes a given protein or polypeptide, e.g., the L1protein or a fragment thereof. In general, such determinations typicallyinvolve comparison of structural features and sequence identities.

As can be seen from the above detail, a vaccine in accordance withcertain embodiments as set forth herein can be either VLPs (virus-likeparticles) or capsomeres containing modified L1 proteins that mayindicate the presence of cross-neutralizing epitopes, in other wordswhich lead to an antibody response directed against various different PVtypes. Such vaccines can be defined as polyvalent vaccines that can beused against infections with various PV types.

As explained above, DNA-free virus capsids, so-called virus-likeparticles (VLP), which accumulate in eukaryotic cells after theexpression of the main structural protein L1, using recombinant vectors,may be suitable for the induction of neutralizing antibodies. VLPs areempty (free of nucleic acid) virus capsids produced using geneticengineering. Substructures of VLPs, too, known as capsomeres, whichresult from an incomplete assembly, for example when modified L1molecules are present, contain neutralizing epitopes, which means theyare suitable for producing vaccines in accordance with the invention.

Epitope is another term for antigen determinants. These are areas on thesurface of an antigen where a specific antibody binds using itsantigen-binding region.

To produce the vaccine according to the invention, a randomly generatedheterologous sequence is inserted into a main structural protein, suchas the L1 gene, of a specific papilloma virus type, especially into thehyper-variable regions of L1 genes.

The expression “insert” in the sense intended in this inventionindicates that the randomly generated heterologous sequences could bepresent in the structural protein gene in addition to the naturallyoccurring epitopes, and/or that the naturally occurring epitope in thegene for the structural protein may be exchanged for a randomlygenerated heterologous sequence.

As an example the production of an L1 gene cassette will be describedbelow, which enables various randomly generated oligo-nucleotides to beinserted into the hyper-variable areas of the L1 structural protein.

For inserting the randomly generated oligo-nucleotides into the DNAsequence of the L1 structural protein, a gene cassette can first beconstructed. This gene cassette is characterized by the fact that, forexample, the DNA code for the hyper-variable areas of the L1 structuralprotein is modified such that silent mutations are used to insertmonovalent interfaces for restriction endonucleases, where the saidinterfaces flank these hyper-variable areas. The term ‘silent mutation’is one used for the introduction of a modified DNA sequence that carriesa recognition point for a specific restriction endonuclease without thischanging the amino-acid sequence. The term ‘monovalent interface’ meansa recognition sequence for a restriction endonuclease that occurs onlyonce in the DNA sequence coding for the target protein. For technicalreasons this has to be a recognition sequence for a restriction enzymethat may not be present additionally in the plasmids employed for theproduction of the variable DNA mixtures. The heterologous, randomlydegenerated oligo-nucleotides may be constructed in such a way that theyare also flanked by the monovalent interfaces, just as they flank thehyper-variable areas of the L1 DNA sequence. This way the gene cassette(in a cloning plasmid) and oligo-nucleotides can be treated with thesame corresponding restriction enzymes. Ligation of theoligo-nucleotides into the gene cassette can then occur.

The expression “heterologous sequences”, as used in this invention,refers to any sort of DNA sequence that differs from the coding DNAsequence for the naturally occurring epitope in the structural proteinin at least one up to a maximum of all nucleotides. This can be achievedby replacing the nucleotides. Because epitopes usually only consist of afew amino-acids that are of the same size range as oligo-proteins, theheterologous sequences can be produced in the customary way, using theDNA sequences of the known epitopes as the basis, for example byoligo-nucleotide synthesis.

For example, if an epitope contains 12 amino acids, the correspondingDNA sequence will consist of 36 nucleotides. For the randomly generatedheterologous sequences, one up to a maximum of all of these nucleotidescan be substituted. Because one nucleotide can be replaced by a total ofthree other nucleotides differing from it, when DNA sequences arerandomly generated a large number, up to several thousand, of new DNAsequences are created that are heterologous to the original DNAsequence. Because the production of this DNA is not site-specific, suchas is the case when for example only one specific nucleotide in a DNAsequence is replaced, it can be defined as a randomly generated DNAsequence

A so-called “random library” is an example of a collection of different,heterologous, randomly generated sequences.

Therefore in the case of the randomly generated heterologous DNAsequences employed to manufacture the vaccine in accordance with theinvention we find they are ones not produced by site-specific mutations;rather, based on known epitope sequences, at least one nucleotide up toa maximum of all nucleotides are replaced in random fashion by one ofthe three other conceivable nucleotides, which gives rise to a randomlygenerated collection of a wide variety of DNA sequences. It isadvantageous if the randomly generated heterologous DNA sequence isoriented to the naturally occurring epitopes in respect of the number ofnucleotides, or ideally has the same number of nucleotides.

When manufacturing the randomly generated heterologous DNA sequences theDNA sequence of the wild type epitope may be obtained again through therandom combining of the nucleotides.

Examples will be given below to show how randomly generatedoligo-nucleotides are obtained. Oligo-nucleotides may be produced usingthe process of oligo-nucleotide synthesis. In this process thenucleotide sequence is produced linearly, in other words the extensionof the chain occurs from the reaction of the already present nucleotidesequence with an active pre-stage of the following nucleotide. But toproduce degenerated oligo-nucleotides not only the activated pre-stageof a nucleotide can be deployed; the activated pre-stages of 2, 3 or 4nucleotides can also be introduced. This gives rise to oligo-nucleotidemixtures coding in this position for several different amino acids. Ifthis process is repeated in subsequent stages of the reaction, acombination of different DNA sequences arises. Here both DNA sequencesthat do not occur in human pathogen PVs result, as well as the sequencesthat code for the wild-type epitope.

Heterologous sequences are inserted into surface loops of L1 capsidsthat are hypervariable among papillomaviruses (Chen et al., Mol Cell 5:,557-567, 2000). As an example, for the main structural protein L1 thesequence area of amino acid 130 to 152 may apply (sequence numbering ofHPV16L1 according to Chen et al., Mol Cell 5:, 557-567, 2000). For the23 amino acids of this sequence section a DNA sequence codes from 69nucleotides. By also introducing the monovalent sequences, DNA sequencesarise with more than 80 nucleotides. Alternatively the area of aminoacids 260-299 or amino acids 349-360 may be selected. With complementaryprimers, different replenishing reactions with DNA polymerases can beused to synthesize the matching strand. Thus, for example, between 3 and200 nucleic acids may be introduced as random sequences; values outsideof this explicitly range are contemplated, as well as all values andranges within the explicitly stated range, e.g, between 6 and 200, atleast 3 or 6, less than 300, and between 12 and 150. The double-strandedDNA obtained this way can then be modified directly with thecorresponding restriction endonucleases and ligated into the L1 genecassette. To obtain greater efficiency the specialist may firstly ligatethe double-stranded DNA sequences, using ‘blunt end’ clonings, intocloning vectors. From these ‘random libraries’ the DNA sequences canthen be re-cloned with a high degree of efficiency into the L1 genecassettes.

The randomly generated heterologous DNA sequences are then, as describedabove, inserted into the gene of the structural proteins of the PV, andin particular into the L1 gene of papilloma viruses of a specific type.HPV, BPV and CRPV are representative types of papilloma viruses.Particularly, insertion into the hyper-variable regions of L1 genes iscarried out. The invention has been described here using the preferredstructural protein gene L1, but it is not limited to this.

The length of the inserted heterologous DNA, in other words the numberof nucleotides, is guided, as described above, by the length of thenaturally occurring epitope. It is selected with particular care beingtaken not to interfere with the formation of the capsomeres and VLPs.Persons of ordinary skill in these arts may use publicly accessibleinformation to identify suitable epitopes of PVs and HPVs. For example,information about HPV epitopes is set forth in Vaccine 2003,21(19-20):2506-15; Intervirology. 2002, 45(1):24-32; Eur J Immunol.2000, 30:2281-9; and Biol Chem. 1999, 380:335-40.

If the original epitopes of the L1 gene are replaced by the randomlygenerated heterologous DNA sequences, it may be that only one of theepitopes is replaced. However several, even all of the maximum possibleepitopes in the L1 gene may be replaced by randomly generatedheterologous sequences.

The L1 gene into which the randomly generated heterologous sequences areinserted may subsequently be cloned into eukaryotic expression vectors.In this case many bacterial clones may arise, and from this large numberof bacteria clones sub-groups can then be formed. In other words thislarge number of bacteria clones is split up into pools of a few thousandbacteria clones and deployed for the production of vaccines inaccordance with the invention.

To produce the vaccine in accordance with the invention one or more thanone expression vector(s) are injected into mammals, whereby thesevectors can be characterized as described above. The term ‘more thanone’ here indicates that a pool of expression vectors can be used,containing up to 10,000, but particularly up to 5,000 expression vectorsthat differ from each other. The differences in the expression vectorsthen exist in particular in the randomly generated heterologous DNAcloned into them. As is clear from the above comments on randomlygenerated heterologous DNA, the expression vectors may also contain DNAsequences that were obtained when randomly generating the DNA sequences,but which—because the generation is also random in this case—areidentical with the DNA sequence for the wild-type epitope. Consequentlyexpression vectors are injected into the mammals where at least some, upto a maximum of all, of them contain randomly generated heterologous DNAsequences inserted into the DNA code.

These preselected pools are used for a DNA vaccination (geneticimmunization). This consists of a recognized immunization procedure inwhich, unlike conventional immunizations, no antigens are injected;instead the DNA code is injected into a corresponding expression vector.The intramuscular application form has been shown to be favorable forDNA vaccination, because obviously in this case absorption andexpression of the gene by the cell takes place before the DNA is brokendown. The immune reaction then takes place in response to the expressedprotein.

One advantage of DNA vaccination can be seen particularly in the factthat the virus particles no longer need to be manufactured and purified,for example by expression of the L1 gene using recombinant vectors. ThusDNA vaccination can be carried out simply and rapidly.

This DNA vaccination may be carried out on mammals such as rats, mice,hamsters and guinea pigs.

Serums can then be obtained from the test animals in the normal way,which are tested for reactivity with different types of papilloma virus.Testing can be done using ELISA, which are specific for papilloma virustypes.

The DNA pools deployed for the DNA vaccination that provoke an immunereaction against different papilloma virus types can subsequently beisolated and again analyzed using DNA. In this way clones can beidentified that code for VLPs or capsomeres and which containcross-neutralizing epitopes. These are epitopes that lead to an antibodyreaction against various papilloma virus types.

After this the corresponding DNA clones can be further examined usingthe customary procedures of genetic engineering, and, as the case maybe, the corresponding virus particles produced, isolated and purified.Additionally L1 molecules, for example, can be expressed, VLPs orcapsomeres can be produced and the immunity of the purified particlescan be examined. Finally it is possible in this way to obtain thevaccine according to the invention, which is characterized by the factthat immunization against more than one papilloma virus type ispossible. The vaccine according to the invention is, therefore, amultivalent vaccine that induces immune protection against diseasescaused by different PV types.

In a preferred embodiment of the vaccine according to the invention thepapilloma virus is a human pathogenic papilloma virus. This makes itpossible to treat diseases caused by human pathogenic papilloma viruseswith the vaccine according to the invention.

In another preferred embodiment the structural protein is L1, becausethis is particularly well suited for producing the vaccine according tothe invention.

In another preferred embodiment the structural protein creates DNA-freevirus capsids or capsomeres.

The object of this invention is also a DNA vaccine comprising one ormore expression vector(s) containing the DNA code for a structuralprotein of papilloma viruses or a fragment thereof, wherein in at leastsome of the expression vectors randomly generated heterologous sequencesare inserted into the DNA code.

As regards the structures and manufacture of the DNA vaccine referenceshould be made to the descriptions given above.

When administering the DNA vaccine according to the invention, thestructural protein gene is expressed and immunization is carried outagainst the expressed protein. In this way immunization is achieved withparticular ease.

An embodiment for manufacture a vaccine is a process wherein

(a) One or more expression vectors are introduced into mammals. Thesevectors encode at least a portion of a structural protein of a papillomavirus (PV), and the expression vectors may further encode at least onerandomly generated heterologous nucleic acid sequence,

(b) sera are obtained from the mammals, and examined for the presence ofat least one polyvalent antibody that recognizes a plurality ofpapilloma virus types,

(c) using the sera, the portions of the expression vectors that encodeepitopes(s) that cause the presence of the at least one polyvalentantibody are identified, and

(d) a vaccine is produced from the expression vectors(s) and/or encodedepitopes thus identified.

The individual steps of the procedure according to the invention havealready been described in connection with the vaccine according to theinvention, so reference is made to the respective embodiments.

The procedure according to the invention is characterized by the factthat the modified genes of the structural protein (insertion of randomlygenerated heterologous DNA) no longer have to be tested individuallybefore immunization for their capacity to form VLPs or capsomeres.Instead, pools of recombinant DNA expression vectors are used toimmunize mammals, in particular mice. The serums obtained are tested forthe presence of antibodies against particles of various papilloma virustypes, especially HPV types. If the reaction is positive, in other wordsif cross-neutralizing epitopes can be demonstrated, the pools ofexpression vectors are isolated and the corresponding proteins analyzed.

This procedure according to the invention enables the testing of a largenumber of variants of papilloma virus particles, especially capsids, fortheir immunogenic qualities, without having to express and purify theparticles individually by expression of the mutated structural proteinbeforehand. Moreover, the procedure according to the invention enablesthe production of highly effective, multivalent papilloma virus vaccinesquickly, simply and at a low cost.

The vaccine according to the invention is best suited as a polyvalentvaccine used in vaccinations against diseases caused by papillomaviruses, particularly diseases that are caused by more than one kind ofpapilloma virus. Examples of these diseases are warts, papillomas,acanthomas, and skin and cervical cancers.

Further examples are provided below. These examples demonstrate that DNAimmunization of animals with a plurality of DNA clones createdmeasurable amounts of antibodies for each clone. Moreover, differentclones may be introduced at ratios of more than 100:1 so that animalsera having antibodies for multiple clones can be readily screened. Infact, ELISA screening techniques were used in the Examples,demonstrating that the proposed methods are compatible withhigh-throughput screening systems. Thus, large libraries can beeffectively analyzed. These examples also demonstrate that DNAimmunization is effective to produce anti-HPV antibodies. Moreover, theanti-HPV antibodies were generated after mice were exposed to aplurality of vectors presented at a variety of ratios relative to eachother. As shown in FIGS. 1-4, anti-HPV 16 L1 antibodies were induced inresponse to a small amount of HPV 16 L1 DNA, even in the presence ofother DNA vectors.

These data show that over one hundred DNA clones may be introduced intoa mouse to generate antibodies against the antigens for which the DNAclones code. The total amount of DNA introduced into the mice tested asshown in FIGS. 1-4 was kept constant. As shown in FIG. 4, the HPV 16 andHPV 18 clones were effective at 100:1 ratios and at 1:100 ratios.Therefore other clones that generate antigens for other types of HPV canalso be expected to be effective at about the same dosage ranges. Sincea plurality of clones are effective at the minimum dose, a multiplicityof clones should be effective at the same dose. Moreover, if about 100clones are introduced at the minimum dose, then the total amount of DNAthereby introduced into an animal would be about the same as what wastested in the Examples. Therefore a single mouse can be inoculated withat least one hundred clones and be expected to produce detectableamounts of antibodies against each clone. Furthermore, all of the dosageranges that were tested and reported herein were found to be effectiveso that higher ratios can reasonably be expected, and the successful useof more than 100 clones in a single animal may reasonably be expected.

EXAMPLE 1

As shown in FIG. 1, mice immunized with various ratios of HPV 16 L1 DNAand an empty pUF3 vector were positive at all tested ratios forantibodies against HPV 16 L1.

DNA of HPV 16 L1 (L1 was cloned into a suitable expression vector) wasmixed in different ratios with other expression constructs (as indicatedin Figures) keeping the total amount of DNA constant. The followingratios were used: 50/0;25/25; 10/40; 5/45; 2.5/47.5; 0.5/49.5; 0/50(HPV16 L1/other vectors as indicated). Mice were then immunized threetimes with 50 μg each time using the ratios above. Sera were collectedand tested in ELISA for antibodies against HPV 16 and 18 L1 virus-likeparticles (produced in baculovirus-infected insect cells). The followingimmunization protocol was used: day 0, 100 μl Cardiotixin i.m.,prebleed; day 5, 50 μg DNA mixture in 100 μl PBS; day 19, 50 μg DNAmixture in 100 μl phosphate buffered saline; day 38, 50 μg DNA mixturein 100 μl PBS; day 48, final bleed. Three mice in each group, mice wereC57/b16 strain. The following ELISA was used: Microtiter plates werecoated with 0.17 μg of VLPs per well. After blocking with skim milk,plates were incubated with antisera (1:25 in skim milk) for 1 hr.Antibodies were detected with goat-anti-mouse IgG horseradish peroxidaseconjugates. After staining, absorption was measured after 20 min. in aTitertech automated plate reader at 405 nm wavelength.

EXAMPLE 2

As shown in FIG. 2, mice immunized with various ratios of HPV 16 L1 DNAand a DNA vector for GFP were positive at all tested ratios forantibodies against HPV 16 L1. Materials and methods as set forth inExample 1 were followed, unless otherwise indicated.

EXAMPLE 3

As shown in FIGS. 3 and 4, mice immunized with various ratios of HPV 16L1 DNA HPV 18 L1 DNA were positive at all tested ratios for antibodiesagainst HPV 16 L1 and HPV 18 L1. Materials and methods as set forth inExample 1 were followed, unless otherwise indicated.

All patents, patent applications, and publications referenced herein arehereby incorporated by reference herein.

1. A method of making an antibody against a plurality of papilloma virustypes, comprising: introducing into a mammal a plurality of DNA clonesfrom a plurality of HPV types, wherein each clone of said plurality ofDNA clones comprises an expression vector comprising DNA that encodes atleast a portion of a L1 structural protein of a human papilloma viruscomprising the coding sequence of at least one naturally occurringepitope of said papilloma virus or at least one randomly generatedheterologous sequence which is derived from the coding sequence of anaturally occurring epitope by the exchange of one or more nucleotides;testing the mammal to detect antibodies against a plurality of papillomavirus types generated by introducing the plurality of DNA clones intothe mammal; and harvesting antibodies from the mammal.
 2. The method ofclaim 1 wherein at least a portion of a structural protein of apapilloma virus comprises at least one randomly generated heterologoussequence which is derived from the coding sequence of a naturallyoccurring epitope by the exchange of one or more nucleotides.
 3. Themethod of claim 2 wherein the DNA clone heterologous sequences have alength of between 6 and 200 bases.
 4. The method of claim 2 wherein theDNA clones comprise DNA that encodes for a hyper-variable portion of theL1 protein, and wherein the at least one randomly generated heterologoussequence comprises a heterologous sequence inserted into thehyper-variable portion of the L1 protein.
 5. The method of claim 2,wherein the least one randomly generated sequence is disposed in asurface loop of the L1 structural protein.
 6. The method of claim 1,wherein the number of DNA clones is between 10 and 10,000.
 7. The methodof claim 1, wherein the number of DNA clones is at least 25.